Research article Received: 3 November 2013,
Revised: 31 December 2013,
Accepted: 9 January 2014
Published online in Wiley Online Library: 20 February 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3151
Quantitative determination of euphol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study Xu Xiea, Yongning Lib, Dongna Gaob, Yu Zhangb and Yanbo Renb* ABSTRACT: Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C18 short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was **) solu-ms2.d
6.5
109.2
6 5.5
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5
95.1 121.2
4.5 4
135.2 191.3
3.5 80.9
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0.5 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
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249.2
393.5
439.4
0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Counts vs. Mass-to-Charge (m/z) +
Figure 2. Product ion mass spectra of [M – H2O + H] of (A) euphol, and (B) oleanic acid (IS).
the two ion-pairs in the range 8–30 V was optimized. Better ion efficiency was obtained at 15 V and 20 V for euphol and IS, respectively. Chromatographic conditions The two compounds were subjected to separation by reversephase Venusil XBP-C18 column (50 × 2.1 mm, 5 μm) using methanol–water–formic acid (95:5:0.1, v/v/v) as the mobile phase at 30 °C. The addition of 0.1% formic acid to the mobile phase could significantly increase the MS intensities of the analyte and IS. Sample preparation
Biomed. Chromatogr. 2014; 28: 1229–1234
The extracted SRM chromatographic profiles are shown in Fig. 3. The retention times of euphol and IS were approximately 1.46 and 0.65 min, respectively. No interference from endogenous substances was observed during the retention of the analyte and IS. In this assay, the intra- and inter-batch precision and accuracy were determined by the replicate analyses of QC samples at three QC levels. The data for precision and accuracy are summarized in Table 1. At each QC level, the intra- and inter-batch precision (RSD) of euphol was in the range 1.28–10.83%. Accuracy was within ±7.04%. These results indicate that the developed method is precise and accurate. The calibration curve exhibited a good linear correlation in the concentration range 27–9000 ng/mL (y = 6.1976 × 10–6x + 2.6793 × 10–4). The coefficient of determination was >0.99. The LLOQ, as the lowest plasma concentration measured with ±20% accuracy and ≤20% precision, was 27 ng/mL. This limit was sufficient for rat pharmacokinetic studies after oral and intravenous administrations of euphol.
Copyright © 2014 John Wiley & Sons, Ltd.
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Different protein precipitation solvents, such as acetonitrile and methanol, were tested. Methanol was used as the optimal solvent because no significant ion suppression effect was observed. Furthermore, this simple protein precipitation method also resulted in high recoveries of euphol and IS with good repeatability.
Method validation
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Figure 3. Selective reaction monitoring chromatograms of (A) blank rat plasma; (B) blank rat plasma sample spiked with euphol at LLOQ and IS; (C) plasma at 1 h after oral administration of euphol (48 mg/kg); and (D) plasma at 1 h after intravenous administration of euphol (6 mg/kg).
Table 1. Results of intra- and inter-batch accuracy and precision Nominal concentration (ng/mL)
Intra-batch Measured concentration (ng/mL)
27 (LLOQ) 90 (LQC) 900 (MQC) 8100 (HQC)
27.52 ± 2.98 93.70 ± 1.24 857.63 ± 12.57 8119 ± 104.08
Inter-batch
Accuracy (RE, %) 1.93 4.11 –4.71 0.23
Precision (RSD, %)
Measured concentration (ng/mL)
10.83 1.32 1.47 1.28
26.66 ± 1.07 91.08 ± 9.23 931.06 ± 30.51 7530 ± 357.62
Accuracy (RE, %) –1.26 1.20 3.45 –7.04
Precision (RSD, %) 4.01 10.13 3.28 4.75
LLOQ, Lower limit of quantification; LQC, low quality control; MQC, medium quality control; HQC, high quality control. Values are given as means ± standard deviation of three determinations (n = 6).
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The extraction recoveries were 93.8% (90 ng/mL), 96.1% (900 ng/mL) and 95.7% (8100 ng/mL) for euphol. The matrix
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effect values were 98.3% (90 ng/mL), 102.8% (900 ng/mL) and 99.3% (8100 ng/mL), respectively.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1229–1234
Quantitative determination of euphol Table 2. Stability of euphol in rat plasma Storage conditions
Euphol Nominal concentration (ng/mL)
Long-term stability
90 900 8100 90 900 8100 90 900 8100 90 900 8100
Short-term stability
Freeze–thaw stability
Preparative stability
Measured concentration (ng/mL)
Accuracy (RE, %)
95.21 ± 5.61 878.57 ± 26.83 7429 ± 69.17 88.37 ± 1.39 936.92 ± 11.08 7647 ± 510.02 87.60 ± 0.98 915.82 ± 67.59 8325 ± 258.61 91.26 ± 4.14 960.22 ± 19.97 7890 ± 480.22
5.79 –2.38 –8.28 –1.81 4.10 –5.59 –2.67 1.76 2.78 1.40 6.69 –2.59
Values are given as means ± standard deviation of three determinations (n = 3).
1400 1200
Concentration (ng/mL)
Table 3. Pharmacokinetic parameters of euphol in rats following intravenous (6 mg/kg) and oral (48 mg/kg) administration
oral group intravenous group
1000
Parameters
Route of dosing Intravenous
800 600 400 200 0 0
4
8
12
16
20
24
Time (h) Figure 4. Mean plasma concentration-time profile of euphol after oral (48 mg/kg) or intravenous (6 mg/kg) administration in rats.
Euphol in plasma was stable at –20 °C for 2 weeks (long-term stability), for 4 h at ambient temperature (short-term stability) and after three freeze–thaw cycles. Euphol was also stable in the autosampler for at least 24 h. The REs were in the range of –8.28–6.69% (Table 2).
Pharmacokinetic study
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1123 ± 221.02 — 4.21 ± 2.03 2788 ± 602.74 2906 ± 676.29 7.85 ± 0.72 2.17 ± 0.53 12.26 ± 4.72 46.01
1110 ± 225.31 4.33 ± 1.03 4.52 ± 1.43 10,172 ± 2566 10,696 ± 3180 4.95 ± 1.72 4.79 ± 1.25 29.80 ± 6.18 —
All data are expressed as means ± standard deviation (n = 6). Cmax, Maximum plasma concentration; Tmax, time to Cmax; t1/2, elimination half-life; AUC, area under the plasma concentration– time curve; MRT, mean residence time; CL, clearance; Vd, volume of distribution; F, absolute bioavailability.
volume of distribution values were estimated at 2.17 ± 0.53 L/(h kg), 7.85 ± 0.72 h and 12.26 ± 4.72 L/kg, respectively. After oral administration at 48 mg/kg, euphol was slowly absorbed and reached a mean Cmax of 1110 ± 225.31 ng/mL at a Tmax of 4.33 ± 1.03 h. The mean AUC0–t and AUC0–∞ values were 10,172 ± 2566 and 10,696 ± 3180 ng h/mL, respectively. The oral F value of euphol was calculated at 46.01% with a t1/2 value of 4.52 ± 1.43 h. The developed LC-MS/MS method is sufficiently sensitive to support further comprehensive pre-clinical and clinical pharmacokinetic studies.
Conclusions In this paper, a simple, rapid and sensitive LC-MS/MS quantitative method was developed and validated to determine euphol concentration in rat plasma for the first time. The method showed excellent selectivity, linearity, precision, and accuracy.
Copyright © 2014 John Wiley & Sons, Ltd.
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The assay method was applied to the pharmacokinetic studies after oral administration of 48 mg/kg or intravenous injection of 6 mg/kg euphol in rats (n = 6). The mean plasma concentrationtime curves are illustrated in Fig. 4, and the major pharmacokinetic parameters of euphol are presented in Table 3. After intravenous administration, the t1/2 value was estimated at 4.21 ± 2.03 h. The mean area under the plasma concentration–time curve from time zero to the last measurable plasma concentration point (AUC0–t) and the mean area under the plasma concentration–time curve from time zero to time infinity (AUC0–∞) values were 2788 ± 602.74 and 2906 ± 676.29 ng h/mL, respectively. Clearance, MRT, and
Cmax (ng/mL) Tmax (h) t1/2 (h) AUC0–t (ng h/mL) AUC0–∞ (ng h/mL) MRT (h) CL/F [L/(h kg)] Vd/F (L/kg) F (%)
Oral
X. Xie et al. The method showed excellent performance because of the following characteristics: small sample volume (50 μL), wide range (27–9000 ng/mL), short running time (only 2.0 min) and a simple preparation process. Furthermore, the proposed method could be successfully applied to pharmacokinetic studies on euphol in rat plasma and on other species or biological matrices.
References Chai YS, Hu J, Wang XK, Wang YG, Xiao XY, Cheng XL, Hua L, Lei F, Xing DM and Du LJ. Euphorbia kansui roots induced-diarrhea in mice correlates with inflammatory response. Chinese Journal of Natural Medicines 2013; 11: 231–239. Chen H, Li Y, Wu X, Li C, Li Q, Qin Z, Yi Y, Chen J, Lai X and Su Z. LC-MS/MS determination of pogostone in rat plasma and its application in pharmacokinetic studies. Biomedical Chromatography 2013; 27: 1092–1099. Cheng XL, Xiao XY, Li GH and Ma SC. HPLC simultaneous determination of euphol and tirucallol in Radix kansui. Chinese Journal of Pharmaceutical Analysis 2009; 29: 1444–1447. Dutra RC, de Souza PR, Bento AF, Marcon R, Bicca MA, Pianowski LF and Calixto JB. Euphol prevents experimental autoimmune encephalomyelitis in mice: Evidence for the underlying mechanisms. Biochemical Pharmacology 2012a; 83: 531–542. Dutra RC, Simão da Silva KA, Bento AF, Marcon R, Paszcuk AF, Meotti FC, Pianowski LF and Calixto JB. Euphol, a tetracyclic triterpene produces antinociceptive effects in inflammatory and neuropathic pain: The involvement of cannabinoid system. Neuropharmacology 2012b; 63: 593–605. Fan Y, Cai DF, Gu XX, Wang GY and Ma J. Treatment of intestinal obstruction with a large dose of kansui. Journal of Emergency in Traditional Chinese Medicine 2005; 14: 278–279. Ge W, Ma B, Yang KH, Zhao FH, Zhang J and Tian JH. Kansui root for treating severe acute pancreatitis: A systematic review. Chinese Journal of Evidence-Based Medicine 2009; 9: 964–968. Guo J, He HP, Fang X, Di YT, Li SL, Zhang Z, Leng Y, Hua HM and Hao XJ. Kansuinone, a novel euphane-type triterpene from Euphorbia kansui. Tetrahedron Letters 2010; 51: 6286–6289. Han YY, Han XY, Zheng JH and Sun JX. The study of Kansui roots and its antifertility effects. Heilongjiang Journal of Traditional Chinese Medicine 2011; 3: 57–59. Hou JJ, Wu WY, Liang J, Yang Z, Long HL, Cai LY, Fang L, Wang DD, Yao S, Liu X, Jiang BH and Guo DA. A single, multi-faceted, enhanced strategy to quantify the chromatographically diverse constituents in the
roots of Euphorbia kansui. Journal of Pharmaceutical and Biomedical Analysis 2014; 88: 321–330. Li XR, Zhang YD, Tang HH and Wu FY. Study of auxiliary therapeutic effect of kansui root on patients with severe acute pancreatitis. China Journal of Modern Medicine 2002; 12: 7–9. Lin MW, Lin AS, Wu DC, Wang SS, Chang FR, Wu YC and Huang YB. Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis. Food and Chemical Toxicology 2012; 50: 4333–4339. Nunomura S, Kitanaka S and Ra C. 3-O-(2,3-dimethylbutanoyl)-13odecanoylingenol from Euphorbia kansui suppresses IgE-mediated mast cell activation. Biological and Pharmaceutical Bulletin 2006; 29: 286–290. Ouyang JB, Deng MY and Ouyang YM. Therapeutic effect on adjuvant treatment of severe acute pancreatitis with Euphorbia kansui Lious. China Journal of Modern Medicine 2004; 14: 96–97. Passos GF, Medeiros R, Marcon R, Nascimento AF, Calixto JB and Pianowski LF. The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice. European Journal of Pharmacology 2013; 698: 413–420. Peng Q, Li GY, Ma YP, Huang J, Wei XY and Wang JH. Chemical constituents of Euphorbia kansui. Biochemical Systematics and Ecology 2012; 43: 64–66. Shen J, Mo X, Tang Y, Zhang L, Pang H, Qian Y, Chen Y, Tao W, Guo S, Shang E, Zhu S, Ding Y, Guo J, Liu P, Su S, Qian D and Duan JA. Analysis of herb-herb interaction when decocting together by using ultra-high-performance liquid chromatography–tandem mass spectrometry and fuzzy chemical identification strategy with polyproportion design. Journal of Chromatography A 2013; 1297: 168–178. The State Pharmacopoeia Commission of the P.R. China. Pharmacopoeia of the P.R. China. Chemical Industry Press: Beijing, 2010; 81–82. US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2001. Available at: http://www. fda.gov/cder/guidance/index.htm Xiu YF, Shi B and Zhu C. Determination of euphol in slices of Euphorbia kansui and Kongxian pills by HPLC-ELSD. Chinese Journal of Experimental Traditional Medical Formulae 2010; 16: 61–63. Yasukawa K, Akihisa T, Yoshida ZY and Takido M. Inhibitory effect of euphol, a triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Journal of Pharmacy and Pharmacology 2000; 52: 119–124. Zheng WF. Study on in vivo antiviral activity of four diterpenoids from ethanol extracts of Euphorbia kansui. Chinese Traditional Herbal Drugs 2004; 35: 65–68.
1234 wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1229–1234
Revised: 31 December 2013,
Accepted: 9 January 2014
Published online in Wiley Online Library: 20 February 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3151
Quantitative determination of euphol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study Xu Xiea, Yongning Lib, Dongna Gaob, Yu Zhangb and Yanbo Renb* ABSTRACT: Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C18 short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was **) solu-ms2.d
6.5
109.2
6 5.5
149.2
5
95.1 121.2
4.5 4
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249.2
393.5
439.4
0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Counts vs. Mass-to-Charge (m/z) +
Figure 2. Product ion mass spectra of [M – H2O + H] of (A) euphol, and (B) oleanic acid (IS).
the two ion-pairs in the range 8–30 V was optimized. Better ion efficiency was obtained at 15 V and 20 V for euphol and IS, respectively. Chromatographic conditions The two compounds were subjected to separation by reversephase Venusil XBP-C18 column (50 × 2.1 mm, 5 μm) using methanol–water–formic acid (95:5:0.1, v/v/v) as the mobile phase at 30 °C. The addition of 0.1% formic acid to the mobile phase could significantly increase the MS intensities of the analyte and IS. Sample preparation
Biomed. Chromatogr. 2014; 28: 1229–1234
The extracted SRM chromatographic profiles are shown in Fig. 3. The retention times of euphol and IS were approximately 1.46 and 0.65 min, respectively. No interference from endogenous substances was observed during the retention of the analyte and IS. In this assay, the intra- and inter-batch precision and accuracy were determined by the replicate analyses of QC samples at three QC levels. The data for precision and accuracy are summarized in Table 1. At each QC level, the intra- and inter-batch precision (RSD) of euphol was in the range 1.28–10.83%. Accuracy was within ±7.04%. These results indicate that the developed method is precise and accurate. The calibration curve exhibited a good linear correlation in the concentration range 27–9000 ng/mL (y = 6.1976 × 10–6x + 2.6793 × 10–4). The coefficient of determination was >0.99. The LLOQ, as the lowest plasma concentration measured with ±20% accuracy and ≤20% precision, was 27 ng/mL. This limit was sufficient for rat pharmacokinetic studies after oral and intravenous administrations of euphol.
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Different protein precipitation solvents, such as acetonitrile and methanol, were tested. Methanol was used as the optimal solvent because no significant ion suppression effect was observed. Furthermore, this simple protein precipitation method also resulted in high recoveries of euphol and IS with good repeatability.
Method validation
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Figure 3. Selective reaction monitoring chromatograms of (A) blank rat plasma; (B) blank rat plasma sample spiked with euphol at LLOQ and IS; (C) plasma at 1 h after oral administration of euphol (48 mg/kg); and (D) plasma at 1 h after intravenous administration of euphol (6 mg/kg).
Table 1. Results of intra- and inter-batch accuracy and precision Nominal concentration (ng/mL)
Intra-batch Measured concentration (ng/mL)
27 (LLOQ) 90 (LQC) 900 (MQC) 8100 (HQC)
27.52 ± 2.98 93.70 ± 1.24 857.63 ± 12.57 8119 ± 104.08
Inter-batch
Accuracy (RE, %) 1.93 4.11 –4.71 0.23
Precision (RSD, %)
Measured concentration (ng/mL)
10.83 1.32 1.47 1.28
26.66 ± 1.07 91.08 ± 9.23 931.06 ± 30.51 7530 ± 357.62
Accuracy (RE, %) –1.26 1.20 3.45 –7.04
Precision (RSD, %) 4.01 10.13 3.28 4.75
LLOQ, Lower limit of quantification; LQC, low quality control; MQC, medium quality control; HQC, high quality control. Values are given as means ± standard deviation of three determinations (n = 6).
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The extraction recoveries were 93.8% (90 ng/mL), 96.1% (900 ng/mL) and 95.7% (8100 ng/mL) for euphol. The matrix
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effect values were 98.3% (90 ng/mL), 102.8% (900 ng/mL) and 99.3% (8100 ng/mL), respectively.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1229–1234
Quantitative determination of euphol Table 2. Stability of euphol in rat plasma Storage conditions
Euphol Nominal concentration (ng/mL)
Long-term stability
90 900 8100 90 900 8100 90 900 8100 90 900 8100
Short-term stability
Freeze–thaw stability
Preparative stability
Measured concentration (ng/mL)
Accuracy (RE, %)
95.21 ± 5.61 878.57 ± 26.83 7429 ± 69.17 88.37 ± 1.39 936.92 ± 11.08 7647 ± 510.02 87.60 ± 0.98 915.82 ± 67.59 8325 ± 258.61 91.26 ± 4.14 960.22 ± 19.97 7890 ± 480.22
5.79 –2.38 –8.28 –1.81 4.10 –5.59 –2.67 1.76 2.78 1.40 6.69 –2.59
Values are given as means ± standard deviation of three determinations (n = 3).
1400 1200
Concentration (ng/mL)
Table 3. Pharmacokinetic parameters of euphol in rats following intravenous (6 mg/kg) and oral (48 mg/kg) administration
oral group intravenous group
1000
Parameters
Route of dosing Intravenous
800 600 400 200 0 0
4
8
12
16
20
24
Time (h) Figure 4. Mean plasma concentration-time profile of euphol after oral (48 mg/kg) or intravenous (6 mg/kg) administration in rats.
Euphol in plasma was stable at –20 °C for 2 weeks (long-term stability), for 4 h at ambient temperature (short-term stability) and after three freeze–thaw cycles. Euphol was also stable in the autosampler for at least 24 h. The REs were in the range of –8.28–6.69% (Table 2).
Pharmacokinetic study
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1123 ± 221.02 — 4.21 ± 2.03 2788 ± 602.74 2906 ± 676.29 7.85 ± 0.72 2.17 ± 0.53 12.26 ± 4.72 46.01
1110 ± 225.31 4.33 ± 1.03 4.52 ± 1.43 10,172 ± 2566 10,696 ± 3180 4.95 ± 1.72 4.79 ± 1.25 29.80 ± 6.18 —
All data are expressed as means ± standard deviation (n = 6). Cmax, Maximum plasma concentration; Tmax, time to Cmax; t1/2, elimination half-life; AUC, area under the plasma concentration– time curve; MRT, mean residence time; CL, clearance; Vd, volume of distribution; F, absolute bioavailability.
volume of distribution values were estimated at 2.17 ± 0.53 L/(h kg), 7.85 ± 0.72 h and 12.26 ± 4.72 L/kg, respectively. After oral administration at 48 mg/kg, euphol was slowly absorbed and reached a mean Cmax of 1110 ± 225.31 ng/mL at a Tmax of 4.33 ± 1.03 h. The mean AUC0–t and AUC0–∞ values were 10,172 ± 2566 and 10,696 ± 3180 ng h/mL, respectively. The oral F value of euphol was calculated at 46.01% with a t1/2 value of 4.52 ± 1.43 h. The developed LC-MS/MS method is sufficiently sensitive to support further comprehensive pre-clinical and clinical pharmacokinetic studies.
Conclusions In this paper, a simple, rapid and sensitive LC-MS/MS quantitative method was developed and validated to determine euphol concentration in rat plasma for the first time. The method showed excellent selectivity, linearity, precision, and accuracy.
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The assay method was applied to the pharmacokinetic studies after oral administration of 48 mg/kg or intravenous injection of 6 mg/kg euphol in rats (n = 6). The mean plasma concentrationtime curves are illustrated in Fig. 4, and the major pharmacokinetic parameters of euphol are presented in Table 3. After intravenous administration, the t1/2 value was estimated at 4.21 ± 2.03 h. The mean area under the plasma concentration–time curve from time zero to the last measurable plasma concentration point (AUC0–t) and the mean area under the plasma concentration–time curve from time zero to time infinity (AUC0–∞) values were 2788 ± 602.74 and 2906 ± 676.29 ng h/mL, respectively. Clearance, MRT, and
Cmax (ng/mL) Tmax (h) t1/2 (h) AUC0–t (ng h/mL) AUC0–∞ (ng h/mL) MRT (h) CL/F [L/(h kg)] Vd/F (L/kg) F (%)
Oral
X. Xie et al. The method showed excellent performance because of the following characteristics: small sample volume (50 μL), wide range (27–9000 ng/mL), short running time (only 2.0 min) and a simple preparation process. Furthermore, the proposed method could be successfully applied to pharmacokinetic studies on euphol in rat plasma and on other species or biological matrices.
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